Application of magnetic field generating device capable of conveniently protecting organ function in assisting in low-temperature keep-alive of in-vitro liver

Abstract

The invention discloses application of a magnetic field generating device capable of conveniently protecting an organ function in assisting in low-temperature keep-alive of an in-vitro liver, and belongs to the technical field of in-vitro organ preservation and magnetic field application. The magnetic field generating device is a square magnet made of neodymium iron boron; the in-vitro liver is located above the magnetic field generating device, the magnetic field intensity of the square magnet is 50-400 mT, and the magnetic field direction is downward. The invention also provides a static cold preservation method of the in-vitro liver organ, which comprises the following steps: placing the liver soaked in the organ preservation liquid at different heights on the surface of the magnetic field generation device, and enabling the liver to be exposed under the magnetic field intensity of 50-400mT to be preserved for 12-48h. The magnetic field generating device has the beneficial effects that the magnetic field generating device has a good promoting effect on static cold preservation of liver organs, reduces oxidative stress of the liver and relieves an ion imbalance state, so that liver cell edema, apoptosis, mitochondrial damage and the like induced by static cold preservation are relieved, and the purposes of prolonging the preservation time and improving the preservation quality are achieved.

Description

Technical Field

[0001] The present invention relates to the technical field of ex vivo organ preservation and magnetic field application, and particularly relates to the application of a magnetic field generating device for conveniently protecting organ function in assisting the hypothermic preservation and viability of an ex vivo liver. Background Art

[0002] Organ transplantation is an important treatment method for end-stage liver diseases. However, among more than 300,000 organ transplantation candidates in China every year, less than 5% of them finally succeed in the transplantation surgery and rehabilitation. The preservation duration and quality of ex vivo organs have a great impact on the transplantation success rate. Due to the lack of precise temperature control and professional preservation and viability techniques, the organ preservation time is extremely short, and irreversible cell damage and protein denaturation are prone to occur. More than 60% of the donated organs are wasted because they cannot be transplanted in time.

[0003] Currently, static cold preservation is still the generally preferred ex vivo organ preservation and viability method in major organ transplantation centers, that is, using a low-temperature environment (2-4 °C) to inhibit cell enzyme activity and metabolic processes, and adding organ preservation solutions (UW solution, CS solution, HTK solution, etc.) to prevent tissue edema and reduce free radicals, etc., so as to achieve the purpose of preserving liver organs. In theory, UW solution can preserve the donor liver for 20-24 hours, but there is cold preservation injury during static cold preservation. The extension of the cold preservation time, that is, the cold ischemia time, will lead to complications, graft failure, and even death of the recipient. The ideal cold preservation time for the donor liver does not exceed 8 hours, and in clinical practice, the preservation time of the donor liver should generally be ≤15 hours. Cold preservation injury refers to mitochondrial injury, ATP energy reduction, cell and tissue edema, and free radical oxidation injury induced by ischemia, hypoxia, and low-temperature environment. Therefore, it is necessary to develop technologies and equipment that can extend the ex vivo organ preservation time and improve the organ preservation quality, so as to gain more time for organ transportation, matching, and surgery. This is an important way to save lives, improve the survival quality of patients, and relieve the burden on families and society, and has extremely high social benefits.

[0004] As a physical field with no radiation, non-invasive, and strong tissue penetration, steady-state magnetic field has been found to participate in regulating cell ion homeostasis and redox processes in recent years. Magnetic fields with specific parameters can reduce liver free radicals induced by alcohol and drugs, and change the Ca 2+ and Na + levels inside and outside cells. Neodymium iron boron permanent magnet is a commonly used rare earth magnet, which has the advantages of being economical, easy to operate, and safe and reliable, and is widely used in electronic products, such as hard disks and mobile phones. There has been no report on applying neodymium iron boron permanent magnet to the field of organ hypothermic preservation and viability to extend the cold preservation time of the liver and improve the organ preservation quality. Summary of the Invention

[0005] The technical problem to be solved by the present invention is how to solve the problems of short preservation time and poor preservation quality existing in the existing ex vivo liver preservation.

[0006] The present invention realizes the solution of the above technical problems through the following technical means:

[0007] In the first aspect of the present invention, an application of a magnetic field generating device for conveniently protecting organ function in assisting low-temperature preservation and survival of ex vivo liver is proposed. The magnetic field generating device is a square magnet made of neodymium iron boron material; the ex vivo liver is located above the magnetic field generating device.

[0008] Preferably, the magnetic field intensity of the square magnet is 50 - 400 mT, and the magnetic field direction is downward.

[0009] Preferably, the length × width × height of the square magnet = 60 mm × 50 mm × 30 mm.

[0010] Preferably, the ex vivo liver is placed in a sterile plastic box filled with organ preservation solution, and the sterile plastic box is located above the magnetic field generating device.

[0011] Preferably, the organ preservation solution is UW (University of Wisconsin) solution or HTK solution (histidine-tryptophan-ketoglutarate solution).

[0012] Preferably, both the magnetic field generating device and the sterile plastic box are in a low-temperature environment, and the low-temperature environment is an ambient temperature of 2 - 4 °C.

[0013] In the second aspect of the present invention, a method for static cold preservation of ex vivo liver organs is proposed, including the following steps:

[0014] Place the liver soaked in the organ preservation solution at different heights on the surface of the magnetic field generating device, so that the liver is exposed to a magnetic field intensity of 50 - 400 mT for 12 - 48 h.

[0015] Preferably, it is exposed to a magnetic field intensity of 50 - 100 mT. More preferably, it is exposed to a magnetic field intensity of 100 mT.

[0016] Preferably, it is preserved for 12 - 36 h.

[0017] Preferably, the liver is a fresh ex vivo liver organ of a mouse.

[0018] The beneficial effects of the present invention are as follows:

[0019] 1. The magnetic field generating device of the present invention has a good promoting effect on the static cold preservation of liver organs, reduces liver oxidative stress and alleviates the state of ion imbalance, thereby reducing liver cell edema, apoptosis and mitochondrial damage induced by static cold preservation, etc., achieving the purpose of extending the preservation time and improving the preservation quality.

[0020] 2. The operation method of the present invention is simple, with the advantages of low energy consumption, low cost and high efficiency. Using the permanent magnet device to assist in the preservation of the liver organ can reduce cold preservation injuries such as hepatocyte edema, apoptosis and oxidative stress, extend the preservation time and improve the preservation quality. The magnetic field generating device of the present invention is also applicable to the static cold preservation of organs such as the heart and kidneys, and can extend the preservation time and improve the preservation quality. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 Schematic diagram of the magnetic field generating device in Embodiment 1 of the present invention;

[0022] Figure 2 Scanning diagram of the magnetic field distribution on the surface of the permanent magnet in Embodiment 1 of the present invention;

[0023] Figure 3 H&E staining diagram of liver tissue sections in Embodiment 2 of the present invention;

[0024] Figure 4 Electron microscopy observation diagram of liver tissue in Embodiment 2 of the present invention;

[0025] Figure 5 Immunohistochemical staining diagram of Ki67 protein in liver tissue and relative quantitative statistical chart of the number of positive cells in Embodiment 2 of the present invention;

[0026] Figure 6 TUNEL staining diagram of liver tissue and quantitative statistical chart of cell apoptosis ratio in Embodiment 2 of the present invention;

[0027] Figure 7 DHE fluorescence imaging diagram of liver tissue and quantitative statistical chart of fluorescence intensity in Embodiment 2 of the present invention;

[0028] Figure 8 Statistical chart of Ca 2+ content in liver tissue in Embodiment 2 of the present invention;

[0029] Figure 9 H&E staining diagram of liver tissue sections in Embodiment 2 of the present invention; DETAILED DESCRIPTION OF THE INVENTION

[0030] To make the objectives, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all of the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0031] The test materials, reagents, etc. used in the following examples can be obtained from commercial sources without special instructions.

[0032] For those not specifying specific techniques or conditions in the examples, they can all be carried out according to the techniques or conditions described in the literature in this field or according to the product instructions. Without special instructions, the quantitative tests in the following examples are all set with more than three repeated experiments, and the results are averaged.

[0033] Example 1: Method for static cold preservation of mouse liver organs assisted by steady magnetic field

[0034] (1) Steady magnetic field device: As Figure 1 shown, this magnetic field device includes a permanent magnet 1, a preservation box 2, an organ preservation solution 3, a mouse liver 4, and a low-temperature environment 5. The permanent magnet 1 is a square magnet made of neodymium iron boron, with the magnetic field direction downward, length × width × height = 60 mm × 50 mm × 30 mm; the preservation box 2 is a sterile plastic box; the organ preservation solution 3 is UW (University of Wisconsin) solution or HTK solution (histidine-tryptophan-ketoglutarate solution); the mouse liver 4 is a complete mouse liver organ obtained by fresh dissection; the low-temperature environment 5 is an environmental temperature of 2 - 4 °C, which can be provided by a 4 °C refrigerator or other low-temperature equipment.

[0035] (2) Acquisition of mouse donor liver: Before the mouse donor liver acquisition experiment, healthy and age-appropriate mice need to be selected, and they are fasted for 12 h but not water-deprived before the operation. Surgical instruments such as scalpels, forceps, and scissors, as well as reagents such as heparinized saline and organ preservation solution, need to be prepared. At the same time, the isoflurane vaporizer in the gas anesthesia device is connected to the anesthesia mask. At the beginning of the experiment, the mouse is first placed in the anesthesia induction box, the isoflurane vaporizer is turned on, the vaporization concentration is set to 3% - 5%, and the oxygen flow rate is adjusted to 0.5 - 1 L / min for induction anesthesia. When the mouse's activity decreases and it is less responsive to stimuli, it is moved to the operating table, and anesthesia is maintained at a concentration of 1.5% - 2.5% through the anesthesia mask and fixed in the supine position. Then, the mouse's abdomen is disinfected from the xiphoid process to the pubic symphysis with iodophor, the skin and abdominal wall are incised along the midline of the abdomen to open the abdominal cavity, the perhepatic ligaments are dissected to fully expose the liver, heparinized saline is slowly perfused through the heart until the liver color becomes lighter, and then the suprahepatic inferior vena cava, infrahepatic inferior vena cava, portal vein and other blood vessels are cut, and the liver is completely removed and quickly placed in the 4 °C organ preservation solution for subsequent experimental use.

[0036] (3) Magnetic field condition treatment: The mouse liver soaked in the preservation solution is placed at different heights on the surface of the permanent magnet and exposed to different magnetic field intensities (50 mT - 400 mT) for 12 - 48 h. As Figure 2 shown, a spatial magnetic field surface magnetic distribution measuring instrument is used to characterize the magnetic field distribution at different heights on the surface of the permanent magnet, and the magnetic fields used are all relatively uniform medium-strength magnetic fields. The average magnetic field intensity on the magnet surface (0 cm) is 400 mT ( Figure 2a); The average magnetic field strength at a distance of 1.6 cm from the surface is 200 mT( Figure 2 b); The average magnetic field strength at a distance of 3.2 cm from the surface is 100 mT( Figure 2 c); The average magnetic field strength at a distance of 6.4 cm from the surface is 50 mT( Figure 2 d).

[0037] (4) Detection of liver structure and function:

[0038] ● H&E staining of liver tissue: Quickly cut the cold-preserved liver tissue into small pieces about 1 cm 3 in size, and immediately fix them in 4% paraformaldehyde fixative for 12 - 24 h; then dehydrate them successively with 70%, 80%, 95%, and 100% alcohol, clear them with xylene, and embed them in paraffin to make paraffin blocks; cut the paraffin blocks into 5-μm-thick sections, stick them on glass slides, and bake the slides at 60 °C for 1 - 2 h; then dewax the sections with xylene successively, hydrate them with alcohol of various grades, stain the nuclei with hematoxylin stain for 5 - 10 min, wash with water, differentiate with 1% hydrochloric acid alcohol for a few seconds and then wash with water again, stain the cytoplasm with eosin stain for 1 - 3 min; finally dehydrate them with gradient alcohol, clear them with xylene, and mount them with neutral gum.

[0039] ● Electron microscopy observation of mitochondria: Cut the cold-preserved liver tissue into small pieces about 1 mm 3 in size, and immediately fix them in pre-cooled electron microscopy fixative for 4 h. Then rinse them 3 times with buffer, 15 min each time. Then dehydrate them with gradient alcohol, from 50%, 70%, 90% to 100% alcohol, 15 min each. Then displace the alcohol with propylene oxide 2 times, 15 min each. Then soak the tissue blocks in a mixture of resin and propylene oxide for 1 h and in pure resin for 3 h. Finally, embed the tissue, section it with an ultramicrotome after polymerization, double-stain it with uranyl acetate and lead citrate, and finally observe the ultrastructure such as liver cell mitochondria under the electron microscope and collect images.

[0040] ● Detection of apoptosis level: The liver tissue fixed in 4% paraformaldehyde is routinely paraffin-embedded and sectioned. After dewaxing the sections to water, according to the method of the apoptosis kit (Beyotime), incubate them with proteinase K working solution at room temperature for 15 - 30 min, and wash them with PBS. Add the TUNEL reaction mixture, incubate them in the dark at 37 °C for 60 min, and wash them with PBS. Then incubate them with converter POD at 37 °C for 30 min, and wash them with PBS. Subsequently, develop the color with DAB substrate, and control the color development time under the microscope. After the color development is completed, counterstain the cell nuclei with hematoxylin for 2 - 5 min, wash with water and turn blue. Finally, dehydrate them with gradient ethanol, clear them with xylene, and mount them with neutral gum, and finally observe the cell apoptosis situation under the microscope.

[0041] ● Proliferation activity detection: Liver tissues fixed with 4% paraformaldehyde were embedded, sectioned, dewaxed, and rehydrated, and antigen retrieval was performed in citrate buffer. Then, the sections were treated with 3% hydrogen peroxide solution for 10 min to eliminate endogenous peroxidase activity, and rinsed with PBS. The primary antibody (Ki67 antibody) was added dropwise and incubated overnight at 4°C. After rinsing with PBS, the secondary antibody was added dropwise and incubated at room temperature for 30 min, followed by another wash with PBS. Then, DAB solution was used for color development, and the cell nuclei were counterstained with hematoxylin, followed by washing with water to turn blue. Finally, dehydration was performed with gradient ethanol, clearing with xylene, and mounting with neutral gum. Finally, the Ki67-positive cells were observed under a microscope.

[0042] ● Oxidative stress level detection: The cryopreserved liver tissues were sectioned at an appropriate thickness. After gently rinsing the sections with PBS buffer, a reactive oxygen species (ROS) fluorescent probe (dihydroethidium, DHE) was added and incubated at 37°C in the dark for 30 min. After incubation, the sections were rinsed 3 times with PBS buffer for 5 min each to remove the unbound probe. An anti-fluorescence quenching mounting agent was added to the sections, and the coverslips were mounted. Finally, observation was performed under a fluorescence microscope, and the fluorescence intensity reflected the ROS level.

[0043] Calcium ion level detection: The Ca 2+ content detection kit (#S1063S, Beyotime) was used to detect the Ca 2+ level in liver tissues. First, the liver tissues were minced and lysed with lysis buffer. Then, centrifugation was performed at 10,000 - 14,000 g for 3 - 5 min at 4°C to obtain the supernatant. Equal amounts of the supernatant were added to a 96-well plate, and 150 μL of the detection working solution was added to each well. After mixing, the samples were incubated at room temperature in the dark for 5 - 10 min. Finally, the OD value at 575 nm was measured with an enzyme-linked immunosorbent assay (ELISA) reader, and the tissue Ca 2+ concentration was calculated.

[0044] Example 2: Method for static cold preservation of mouse liver organs

[0045] (1) Selection and preparation of experimental animals: Healthy mice with appropriate body weight were selected. Before the experiment, the mice were fasted, but sufficient water was provided to reduce the interference of gastrointestinal contents on surgical operations. At the same time, surgical instruments such as ophthalmic forceps and scissors were prepared, and the instruments were ensured to be sterile.

[0046] (2) Liver acquisition: The mice were anesthetized with isoflurane gas. In a sterile environment, the abdominal cavity of the mice was opened. Method 1: Heparinized normal saline was slowly perfused through the heart until the liver color became lighter, and then the superior vena cava, inferior vena cava, and portal vein were cut, and the liver was removed intact and quickly placed in a 4°C organ preservation solution (UW solution or HTK solution). Method 2: The intact liver of the mice was directly dissected and the excised liver was perfused with a 4°C pre-cooled organ preservation solution.

[0047] (3) Static cold preservation: After irrigation, completely immerse the liver in the preservation solution, seal the container, and place it in a refrigerator at 4°C or a cryopreservation device. During preservation, gently shake the container at regular intervals to ensure uniform distribution of the preservation solution. Meanwhile, liver tissue sections can be observed under a microscope to monitor the morphological changes of hepatocytes and evaluate the preservation effect. The organ preservation duration corresponds to the treatment time (12 - 48 h) of the magnetic field-assisted preservation experiment.

[0048] (4) Detection of liver structure and function: The same as in Example 1.

[0049] (5) Analysis of experimental results:

[0050] As Figure 3 shown, mouse ex vivo livers after cold preservation were treated with magnetic fields of different intensities (50 - 400 mT) for 12 h. After the experiment, liver tissues were collected for H&E staining to evaluate the pathological structural changes of the liver tissue. The results showed that compared with fresh liver tissue, obvious cellular edema occurred in the liver tissue of the cold preservation control group, accompanied by phenomena such as nuclear shrinkage and deepening of staining. However, the liver injury in the magnetic field treatment group was significantly alleviated, especially the cellular edema was significantly reduced.

[0051] As Figure 4 shown, further, electron microscopy observations of the microstructure of cold-preserved livers were performed to evaluate the magnetic field preservation effect. The results showed that the mitochondrial structure of the cold preservation control group liver was damaged and the electron density decreased, while the endoplasmic reticulum (indicated by red arrows) and mitochondria (indicated by black arrows) of the liver in the magnetic field treatment group were less damaged, and the mitochondrial cristae and double membrane structure were relatively intact. Moreover, the regulatory effect of the 50 - 100 mT magnetic field was more significant.

[0052] As Figure 5 shown, immunohistochemical experiments of the proliferation molecule protein Ki67 were performed on liver tissue sections. The liver has extremely strong regenerative ability, which is manifested as the activation of cell proliferation and division when the liver is severely damaged. The results showed that the cold preservation process had no significant effect on hepatocyte proliferation, but the 400 mT magnetic field group showed enhanced liver proliferation activity, which was consistent with the literature reports that downward magnetic fields can promote DNA synthesis and enhance the proliferation activity of liver cells.

[0053] As Figure 6 shown, apoptosis is also one of the important indicators for evaluating liver injury. The TUNEL experiment found that static cold preservation could cause obvious apoptosis in liver tissue. However, magnetic field-assisted liver cold preservation with 50 - 400 mT could effectively reduce the level of liver cell apoptosis, thereby protecting the function of the liver organ.

[0054] As Figure 7As shown, DHE staining was performed on liver tissue sections to evaluate the level of reactive oxygen species (ROS). Liver oxidative stress is a common phenomenon during static cold preservation. Therefore, reducing oxidative stress is an important way to alleviate liver cold preservation injury. The results showed that magnetic field treatment could significantly reduce the ROS level in liver tissue, and the magnetic field of 100 - 200 mT had a better effect.

[0055] As Figure 8 shown, Ca 2+ level detection was performed on liver tissue to evaluate the changes in liver ion homeostasis. Hypothermia, ischemia, and hypoxia during static cold preservation can lead to ion imbalance in liver cells. In particular, cell edema and mitochondrial damage are both related to intracellular Ca 2+ overload. The experimental results showed that compared with the cold preservation control group, the Ca 2+ level in the magnetic field group was significantly reduced. Moreover, the reduction effect of the 50 - 100 mT magnetic field group was better than that of the 200 - 400 mT group.

[0056] As Figure 9 shown, the livers of cold - preserved mice were treated with a 400 mT magnetic field for 12 h, 24 h, 36 h, and 48 h, and H&E staining was performed on liver tissue sections to evaluate the effect of the magnetic field on the preservation duration. The results showed that compared with the cold preservation control group, the liver cold preservation injury in the magnetic field group was alleviated; moreover, within 12 - 36 h, the 400 mT magnetic field had a good protective effect on liver cold preservation.

[0057] The above embodiments are only used to illustrate the technical solutions of the present invention, not to limit it; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that they can still modify the technical solutions described in the foregoing embodiments, or perform equivalent replacements for some of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. Application of a magnetic field generating device for conveniently protecting organ function in assisting cryopreservation of ex vivo liver, characterized in that: The magnetic field generating device is a square magnet made of neodymium iron boron material; the isolated liver is located above the magnetic field generating device.

2. The use according to claim 1, characterized in that: The magnetic field strength of the square magnet is 50-400 mT, and the magnetic field direction is downward.

3. The use according to claim 1, characterized in that: The length×width×height of the square magnet=60mm×50mm×30mm.

4. The use according to claim 1, characterized in that: The isolated liver is placed in a sterile plastic box filled with organ preservation fluid, and the sterile plastic box is located above the magnetic field generating device.

5. The use according to claim 1, characterized in that: The organ preservation solution is UW solution or HTK solution.

6. The use according to claim 1, characterized in that: The magnetic field generating device and the sterile plastic box are both in a low temperature environment, and the low temperature environment is an ambient temperature of 2 to 4°C.

7. A method for static cold preservation of an ex vivo liver organ, characterized in that: The following steps are involved: The liver soaked in the organ preservation solution is placed on the surface of the magnetic field generating device at different heights, so that the liver is exposed to a magnetic field strength of 50-400mT and preserved for 12-48h.

8. The method according to claim 7, characterized in that Exposure to magnetic field strength of 50-100mT.

9. The method according to claim 7, characterized in that: Store for 12-36 hours.

10. The method according to claim 7, characterized in that The liver is a fresh isolated mouse liver organ.

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